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The CONffIDENCE project - The outcome of WP3 on heavy metals
Sloth, Jens Jørgen
Publication date:2012
Document VersionPublisher's PDF, also known as Version of record
Link back to DTU Orbit
Citation (APA):Sloth, J. J. (Author). (2012). The CONffIDENCE project - The outcome of WP3 on heavy metals. Sound/Visualproduction (digital)
Work package 3 Heavy metals CONffIDENCE Stakeholder workshop Brussels 20. September 2012
www.conffidence.eu
WP leader Jens J. Sloth
Agenda The CONffIDENCE project – general information
WP3 on ”heavy metals” – in focus
Inorganic arsenic - SPE HG-AAS method - seafood samples - rice samples
Methylmercury - HPLC-ICPMS method - seafood samples - feed samples
Method development
Surveys Risk-benefit
analysis
Method validation
In-house and ILC
CONffIDENCE in a nutshell
CONtaminants in Food and Feed – Inexpensive DEtectioN for Control of Exposure
Collaborative Project: FP7 (European Commission) Duration: May 2008 – Dec 2012 16 partners from 10 countries, representing universities,
research institutes, industry and SMEs Budget: 7.5 Mio € Coordinator: RIKILT - Institute of Food Safety, part of
Wageningen UR (NL) WP3 leader: DTU Food
The commodities
Food & Feed Fish/shellfish and fish feed Cereals and cereal-based feed Potatoes/vegetables Honey Eggs Meat Dairy products
The target contaminants POPs: - dioxin-like PCBs + metabolites - brominated flame retardants - polycyclic aromatic hydrocarbons (PAH) Perfluorinated compounds (PFCs) Pesticides: paraquat/diquat, dithiocabamates Veterinary drugs: - antibiotics, e.g. tetracyclines - coccidiostats, e.g. ionophores Heavy metals speciation: -inorganic arsenic -methylmercury Biotoxins: - alkaloids - marine biotoxins - mycotoxins
www.conffidence.eu
Newsletter – 2 times/year
WP3 overall objectives
Objectives Development of simplified methodologies for the determination of
1) inorganic arsenic (iAs) in seafood
2) methylmercury (MeHg) in marine based food and feed.
2 parallel approaches were followed 1) cytosensor approach using luminescent bacterial cell biosensors (CYT)
2) solid phase extraction approach followed by AAS (SPE-AAS)
WP3 - relevance Current situation in EU legislation:
Foodstuffs MLs for Pb, Cd, Hg and Sn EU directive 2006/1881/EC (and amendments)
Animal feedingstuffs MLs for As, Pb, Cd and Hg EU directive 2002/32/EC (and amendments)
Only maximum levels for total concentration of the metals
Arsenic inorganic As (iAs) is the toxic form of As Lack of specific data on iAs (EFSA, 2009 and JECFA, 2010) Lack of validated, standardised methods (EFSA, JECFA)
Mercury Methylmercury is considered more toxic than inorganic Hg (iHg)
Seafood/marine feed Seafood is the predominant source of As and Hg in the European diet Focus on marine feed and food sample types
EFSA (2009) and JECFA (2010) opinions on arsenic in food
Old PTWI value (WHO, 1988) was withdrawn NEW! BMDL1.0 = 0.3 – 8 µg/kg bw per day for inorganic arsenic => EU dietary exposures within this range => Risk to some consumers cannot be excluded NEW! BMDL0.5 = 3 µg/kg bw per day for inorganic arsenic => 0.5% increased incidence of lung cancer for 12 y exposure “…there is a need to produce speciation data for different food commodities
to support dietary exposure assessment…”
“…more accurate information on the inorganic arsenic content of foods is needed to improve assessments of dietary exposures to inorganic arsenic”
“…need for validated methods for selective determination of inorganic
arsenic in food matrices”
hydride generation atomic absorption
spectrometry
Arsenic speciation analysis speciation alternative: SPE, HG-AAS
inorganic arsenic
As
O
OHOH
OH-
µ-wave extraction
SPE separation
HG-AAS detection
µ-wave extraction - oxidation of As(III) to As(V)
µ-wave oven
0.2 g sample + 10 mL extractant
(0.06 M HCl, 3% H2O2)
Glas vessel
25 minutes at 90°C
Centrifugation 10 min 2100 x g
centrifuge
SPE protocol - separation of As species
Strong anion exchange SPE column
silica based Strata SAX
500 mg/6 mL, Phenomenex
As
O
OHOH
OH-
The charge of the arsenic species depends on pH
@ pH = 6 iAs(V) is negatively charged
Sequential elution Separation of inorganic As from organo As species by SPE
SPE protocol - Separation of As species
Wash 0.5 M CH3COOH
Load Buffered sample: pH 5.0-7.5
Elute 0.5 M HCl
Condition 100 % MeOH
Equilibrate Buffer: 20mM (NH4)2CO3, 0.03 M HCl and 1.5% H2O2
As
O
OHOH
OH-
µ-wave extraction
Separation by SPE
Detection by HG-AAS
SPE-HG-AAS – a novel speciation alternative…
Sequential elution for selective off-line separation of inorg As from organo As species by SPE
OrganoAs compounds
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.500
50001000015000200002500030000350004000045000500005500060000650007000075000
Time-->
Abundance
Ion 75.00 (74.70 to 75.70): 53.DIon 75.00 (74.70 to 75.70): 54.D (*)Ion 75.00 (74.70 to 75.70): 63.D (*)Ion 75.00 (74.70 to 75.70): 64.D (*)
Sample eluate inorganic As
Sample load
Wash Inorganic As
OrganoAs
load wash elute
HPLC-ICPMS of SPE fractions
Inorganic arsenic: SPE-HG-AAS versus HPLC-ICP-MS
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 0.5 1 1.5 2 2.5 3
ICP
(mg/
kg)
AAS (mg/kg)
fdkl
The detection methods were not significantly different (t Test, 95% confidence)
Inorganic arsenic 72 marine extracts
blank, spiked and natural incurred samples
Spike low
Spike medium
Spike high
TORT-2 DORM-3
iAs level (mg/kg) 0.5 1 1.5 0.9* 0.2* Observations (N) 9 9 9 6 6 Mean recovery (%) 101 103 104 100 90 Repeatability RSDr (%) 4 8 5 3 7 Reproducibility RSDIR (%) 5 9 6 9 13 Horwitz Rel. Std. (%) 18 16 15 16 20 *Reference value determined by HPLC-ICP-MS
Setup Spiked samples Trout, oyster Natural incurred samples TORT-2, DORM-3 Analysed in triplicates on 3 different days 2 technicians
In-house validation – iAs by SPE-HGAAS
Results overview 0.08 mg/kg limit of detection (LOD) 3-8% repeatability 5-13% reproducibility 90-104% recovery
Rasmussen et al, ABC 2012
Collaborative trial – marine samples
Sample Description ~conc level (mg/kg) WP3-2 IMEP32-4 fish meal spiked 1 WP3-3 IMEP32-5 fish fillet spiked 2.5 WP3-4 Blue mussel powder 0.3 WP3-5 Crab powder 0.1 WP3-6 DORM-3 Dogfish muscle 0.2 WP3-7 TORT-2 Lobster Hepatopancreas 0.8
- 10 labs (one lab gave 2 sets of results => 11 datasets) - SPE separation procedure was followed - Both HG-AAS and ICPMS were used for determination of iAs
Collaborative trial – marine samples Unit WP3-2 WP3-3 WP3-4 WP3-5 WP3-6 WP3-7 WP3-9 No of labs 11 11 11 11 11 11 11 No of non-compliant labs 3 2 6 1 1 3 2 No of compliant labs 8 9 5 10 10 8 9 Overall mean mg kg-1 1,03 2,57 0,26 0,14 0,19 0,76 0,16
Sr mg kg-1 0,12 0,20 0,04 0,03 0,02 0,06 0,03 RSDr % 11,5 7,9 14,1 23,2 13,1 7,6 18,3
rL mg kg-1 0,33 0,57 0,10 0,09 0,07 0,16 0,08
SR mg kg-1 0,17 0,34 0,07 0,09 0,04 0,13 0,05 RSDR % 16,5 13,4 26,7 64,1 22,1 17,4 30,0
RL mg kg-1 0,47 0,96 0,19 0,26 0,12 0,37 0,13 Horwitz value 15,8 13,8 19,5 21,3 20,4 16,6 21,0 HorRat 1,0 1,0 1,4 3,0 1,1 1,1 1,4
- Precision: RSDr : 8 - 14% and RSDR :13 - 27% - Accuracy: 89-100% - Measurement range: 0.2 - 2.6 mg/kg - HorRat: 1.0 – 1.4 - HG-AAS vs ICPMS: no difference
- Blue mussel sample (WP3-4): not satisfactory results
Survey data – marine samples
Inorganic arsenic - 148 seafood samples - all fish <0.04 mg/kg - bivalves <0.01 – 0.07 mg/kg
Atlantic halibut (Hippoglossus hippoglossus)Cod (Gadus morhua)Greenland halibut (Rheinhardtius hippoglossoides)Mackerel (Scomber scombrus)North Sea herring (Clupea harengus)NSS herring (Clupea harengus)Tusk (Brosme brosme)
Inorganic arsenic in wild caught fish => no concern
Norwegian survey 900 individual fish samples Atlantic halibut Cod Greenland halibut Mackerel Herring Tusk
Results Total arsenic………..0.3-110 mg/kg Inorganic arsenic…. < 0.01 mg/kg
(only 37 samples > LOQ)
Julshamn and Sloth, Fd Addit Contam B, 2012, in press
y = 0.51x - 1.51R2 = 0.9146
0
1
2
3
4
5
6
7
0 2 4 6 8 10 12 14 16
Total As / mg kg-1
Inor
gani
c A
s / m
g kg
-1
Sloth and Julshamn, 2008, J. Agri.Food Chem., 56, 1269-1273
Data from 175 blue mussel (Mytilus edulis) samples collected along the Norwegian Coastline.
NORWAY
Total As = 13.8 mg/kg Inorg As = 5.8 mg/kg Fraction = 42 %
...but in bivalves high contents in some samples...
90℃ waterbath,1h Sample + 10 mL extractant (0,1 M HNO3, 3% H2O2) centrifugation
SPE HG-AAS – iAs in rice
SPE separation
HG-AAS
SPE HG-AAS – iAs in rice - validation
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
1,00
0,00 0,20 0,40 0,60 0,80 1,00
HPL
C-IC
P-M
S (m
g/kg
)
SPE HG-AAS (mg/kg)
SPE HG-AAS vs HPLC-ICPMS
Accuracy Target value (mg/kg) Found (mg/kg)mean +/- 2s
IMEP-107 0,107 +/- 0,014 0,108 +/- 0,017 (N=6)NIST1586a 0,097 0,101 +/- 0,014 (N=6)
Spiked samples0,30 mg/kg 105 % (N=9)0,55 mg/kg 106 % (N=9)0,80 mg/kg 106 % (N=9)
Spike level Recovery
PrecisionRepeatability RSDr 4,8 %Reproducibility RSDR 7,8 %
LoD / LoQLoD (k=3) 0,02 mg/kgLoQ (k=6) 0,04 mg/kg
SPE HG-AAS – iAs in rice – collaborative study
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
0 1 2 3 4 5 6 7 8 9 10 11
X1
X2
X3
X4
Mean value
WP3-9 No of labs 11 No of non-compliant labs 2 No of compliant labs 9 Overall mean 0,16 Sr 0,03 RSDr 18,3 rL 0,08 SR 0,05 RSDR 30,0 RL 0,13 Horwitz value 21 HorRat 1,4
Test sample: Wholemeal rice flour (organic)
Survey data – iAs in rice samples
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
1 2 3 4 5 6 7
iAs
cont
ent (
mg/
kg)
White rice types
Brown rice types
Rice crackers
21 samples (so far) White rice Brown rice types Rice crackers
Future ML ?
HPLC Column ICPMS Result
Speciation analysis of mercury by HPLC-ICPMS
Ultra- sonification
0.5 gram sample (2 x extraction with 5 ml 5 M HCl)
Centrifugation
pH adjustment
Cation exchange (Hamilton PRP X200 SCX)
HPLC-ICPMS
0
1000
2000
3000
4000
5000
6000
7000
0 2 4 6 8 10 12
Siga
nl in
tens
ity (c
ps 20
2 Hg)
Time (min)
HPLC-ICPMS chromatogram of DORM-3 (Dogfish muscle)
Cation exchange HPLC-ICPMS
iHg
MeHg
Performance of the HPLC-ICP-MS method for determination of MeHg
DORM-2 Dogfish
TORT-2 Lobster
DORM-3 Dogfish
Fishfeed #1 Fishfeed#2 Codfish Salmon
Ref level (mg/kg) 4.47 0.15 0.36 0.21 0.06 0.17 0.06 Observations (N) 9 15 9 9 9 9 9 Mean recovery (%) 94 102 96 - - - - Repeatability RSDr (%) 3 4 3 11 13 5 13 Reproducibility RSDIR (%) 8 12 8 11 15 12 20 Horwitz Rel. Std. (%) 13 21 19 20 25 21 25
Setup Natural incurred samples - CRMs (DORM-2, DORM-3 and TORT-2) - fish feed, codfish and salmon Analysed in triplicates on 3 different days 2 technicians Results overview
0.004 mg/kg limit of detection (LOD) Mean repeatability = 7% Reproducibility < Horwitz RSD 94-102% recovery
Collaborative trial – marine samples
Target value LAB1 LAB2 LAB3 LAB4
WP3-1 Complete feed (spiked) 0,19 0,21 0,20 WP3-3 Fish fillet (spiked) 1,8 2,08 1,91 WP3-5 Crab powder 0,28 0,35 0,34 WP3-6 DORM-3 0,355 0,38 0,34 WP3-7 TORT-2 0,152 0,17 0,15 WP3-8 CE464 Tunafish 5,5 5,53 5,61
- Small scale ILC (4 labs) - 6 samples (0,15 – 5,5 mg/kg) - Both seafood and feed
Data to be produced Sept/Oct
Survey data - MeHg in fish feed and ingredients Type Sample
ID % Fat Hg (total)
(µg/kg) MeHg
(µg/kg) Fish silage 204557 11.8 39 <30
205398 11.3 40 <30 207967 10.7 39 <30 207976 9.2 11 <30 208547 11.3 55 <30
Fish oil 201224 100 <10 na 201225 100 <10 na 205376 100 <10 na
Complete feed 207847 34.6 24 <30 210554 28.8 18 <30 210555 17.0 36 <30 210606 24.8 49 32
Fish meal 201226 13.7 120 125 201227 14.0 93 79 202128 13.7 71 45 202141 8.2 48 30 204687 12.0 30 <30 204836 10.3 43 <30 206945 10.4 34 <30 207833 12.0 33 <30 207899 12.3 27 <30 210705 11.0 69 53 211035 6.0 67 55 211612 7.9 40 <30 211662 14.4 61 53 211669 9.7 44 32
All samples collected as part of the national
surveillance/feed-control programme in Denmark
EU maximum level -No ML for MeHg
- 0.2 mg/kg for total Hg (2010) (before 2010 the ML= 0.1 mg/kg)
-all samples < ML
Survey data – MeHg in seafood
Whiting
bivalves pangasius
salmon
Cod
Herring
Hake
tuna
Output from CONffIDENCE WP3 Methods: - iAs in marine samples by SPE HG-AAS - iAs in rice samples by SPE HG-AAS - MeHg in marine samples by HPLC-ICPMS Collaborative trials: - iAs in marine samples by SPE HG-AAS (10 labs) - MeHg in marine samples by HPLC-ICPMS (4 labs) - ”target values” established for future QA purposes Survey data: - iAs in marine samples (N=130) - iAs in rice samples (N=30) - MeHg in marine samples (N=130) Contribution to risk-benefit analysis : - Seafood samples analysed for POPs and fatty acids (with WP1) - Reported to EFSA databases for future risk evaluations
Further information
www.conffidence.eu CONffIDENCE newsletters Scientific publications Hedegaard and Sloth, Heavy metal speciation in feed: why and how?, BASE, 2011, 15, 45-51. Rasmussen et al, Development and validation of an SPE HG-AAS method for determination of inorganic arsenic in
samples of marine origin, Anal Bioanal Chem, 2012, 403, 2825-2834. Rasmussen et al, Development and validation of a HPLC-ICPMS method for determination of methylmercury in
marine food and feed, Anal Bioanal Chem (CONffIDENCE special issue), in prep (expected 2013) Sloth et al, Contaminant and fatty acid profiles in European seafood, in prep (expected 2013)
Contact: Jens J. Sloth ([email protected]) (WP3 leader)
Thanks for your attention!